1. Field of the Invention
The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to seamlessly cloning or subcloning one or more nucleic acid molecules. The present invention also relates to seamless cloning of nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using the methods of the present invention.
2. Related Art
A significant problem with many of the currently available molecular cloning techniques results from the reliance upon restriction sites. These techniques result in the presence of extraneous polynucleotides in the amplification products even after restriction digestions. Such extraneous polynucleotides can introduce design limitations on the cloned product which often interfere with the structure and function of the desired gene products, be they RNA, DNA or protein.
One method of joining nucleic acids without introducing extraneous bases or relying on the presence of restriction sites is splice overlap extension (SOE) (Yon et al, Nucl. Acids Res. 17:4895 (1989) and Horton et al., Gene 77:61-68 (1989)). This method is based on the hybridization of homologous 3′ single-stranded overhangs to prime synthesis of DNA using each complementary strand as a template. Although this technique can join fragments without introducing extraneous nucleotides (in other words, seamlessly), it does not permit the easy insertion of a DNA segment into a specific location when seamless junctions at both ends of the segment are required. Nor does this technique allow for joining fragments with a vector. Ligation with a vector must be subsequently performed by incorporating restriction sites onto the termini of the final SOE fragment. Finally, this technique is particularly awkward when trying to exchange polynucleotides encoding various domains or mutation sites between genetic constructs encoding related proteins.
Sorge et al., U.S. Pat. No. 6,261,797 describe a method by which polynucleotide sequences of interest are synthesized using one or more synthesis primers, wherein at least one of the primers is a releasable primer. After synthesis, the synthesis product is cleaved by a releasing enzyme. The releasable primers of Sorge et al. comprise a recognition site for a type IIs restriction endonuclease, principally Eam1105I. This then allows for “seamless domain replacement” where synthesis reactions allow the production of a polynucleotide of interest by synthesizing two different polynucleotide sequences using separate sets of primers, cleaving the synthesis products with a releasing enzyme, and ligating together the two sets of release synthesis products.
Type IIs Restriction Enzymes
Restriction enzymes can be grouped based on similar characteristics. In general there are three major types or classes: I, II (including IIs) and III. Class I enzymes cut at a somewhat random site from the enzyme recognition sites (see Old and Primrose, Principles of Gene Manipulation, Blackwell Sciences, Inc., Cambridge, Mass., (1994)). Most enzymes used in molecular biology are type II enzymes. These enzymes recognize a particular target sequence (i.e., restriction endonuclease recognition site) and break the polynucleotide chains within or near to the recognition site. The type II recognition sequences are continuous or interrupted. Class IIs enzymes (i.e., type IIs enzymes) have asymmetric recognition sequences. Cleavage occurs at a distance from the recognition site. These enzymes have been reviewed by Szybalski et al. Gene 100:13-26 (1991). Class III restriction enzymes are rare and are not commonly used in molecular biology.
Type-IIs endonucleases generally recognize non-palindromic sequences and cleave outside of their recognition site, thus producing overhangs of ambiguous base pairs. (Szybalski, Gene 40:169-173 (1985).) Additionally, as a result of their non-palindromic recognition sequences, the use of type-IIs endonucleases will generate more markers per kB than a similar type-II endonuclease, e.g., approximately twice as often. U.S. Pat. No. 4,293,652 discloses a linker with a type-IIs enzyme recognition sequence to permit synthesized DNA to be inserted into a vector without disturbing a recognition sequence. Brousseau et al. (Gene 17:279-289 (1982)) and Urdea et al. (Proc. Natl. Acad. Sci. USA 80:7461-7465 (1983)) disclose the use of type-IIs enzymes for the production of vectors to produce recombinant insulin and epidermal growth factor respectively.
Thus, there remains a need in the art for methods and compositions that allow for insertion of nucleic acid molecules into specific locations of other nucleic acid molecules with seamless junctions at one or both ends. There is also a need in the art for methods and compositions that allow for transfer of these seamlessly cloned sections from one nucleic acid molecule to another. The present invention fulfills these needs.